Method of treating hyperproliferative vascular disease

ABSTRACT

This invention provides a method of preventing or treating hyperproliferative vascular disease in a mammal by administering an antiproliferative effective amount of rapamycin alone or in combination with mycophenolic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/238,305 filed May 12,1994, now U.S. Pat. No. 5,561,781 which is a continuation-in-part ofU.S. Ser. No. 07/980,000 filed Nov. 23, 1992, now abandoned which is acontinuation of U.S. Ser. No. 07/819,314 filed Jan. 9, 1992 nowabandoned.

BACKGROUND OF THE INVENTION

Many individuals suffer from heart disease caused by a partial blockageof the blood vessels that supply the heart with nutrients. More severeblockage of blood vessels in such individuals often leads tohypertension, ischemic injury, stroke, or myocardial infarction.Typically vascular occlusion is preceded by vascular stenosis resultingfrom intimal smooth muscle cell hyperplasia. The underlying cause of theintimal smooth muscle cell hyperplasia is vascular smooth muscle injuryand disruption of the integrity of the endothelial lining. The overalldisease process can be termed a hyperproliferative vascular diseasebecause of the etiology of the disease process. Intimal thickeningfollowing arterial injury can be divided into three sequential steps: 1)initiation of smooth muscle cell proliferation following vascularinjury, 2) smooth muscle cell migration to the intima, and 3) furtherproliferation of smooth muscle cells in the intima with deposition ofmatrix. Investigations of the pathogenesis of intimal thickening haveshown that, following arterial injury, platelets, endothelial cells,macrophages and smooth muscle cells release paracrine and autocrinegrowth factors (such as platelet derived growth factor, epidermal growthfactor, insulin-like growth factor, and transforming growth factor) andcytokines that result in the smooth muscle cell proliferation andmigration. T-cells and macrophages also migrate into the neointima.[Haudenschild, C., Lab. Invest. 41:407 (1979); Clowes, A., Circ. Res.56:139 (1985); Clowes, A., J, Cardiovas. Pharm. 14 (Suppl. 6): S12(1989); Manderson, J., Arterio. 9:289 (1989); Forrester, J., J. Am.Coll. Cardiol. 17:758 (1991)]. This cascade of events is not limited toarterial injury, but also occurs following injury to veins andarterioles.

Vascular injury causing intimal thickening can be broadly categorized asbeing either biologically or mechanically induced. Artherosclerosis isone of the most commonly occurring forms of biologically mediatedvascular injury leading to stenosis. The migration and proliferation ofvascular smooth muscle plays a crucial role in the pathogenisis ofartherosclerosis. Artherosclerotic lesions include massive accumulationof lipid laden "foam cells" derived from monocyte/macrophage and smoothmuscle cells. Formation of "foam cell" regions is associated with abreech of endothelial integrity and basal lamina destruction. Triggeredby these events, restenosis is produced by a rapid and selectiveproliferation of vascular smooth muscle cells with increased new basallamina (extracellular matrix) formation and results in eventual blockingof arterial pathways. [Davies, P. F., Artherosclerosis Lab. Invest. 55:5(1986)].

Mechanical injuries leading to intimal thickening result followingballoon angioplasty, vascular surgery, transplantation surgery, andother similar invasive processes that disrupt vascular integrity.Intimal thickening following balloon catheter injury has been studied inanimals as a model for arterial restenosis that occurs in human patientsfollowing balloon angioplasty. Clowes, Ferns, Reidy and others haveshown that deendothelilization with an intraarterial catheter thatdilates an artery injures the innermost layers of medial smooth muscleand may even kill some of the innermost cells. [Schwartz, S. M., HumanPathology 18:240 (1987); Fingerle, J., Ateriosclerosis 10:1082 (1990)]Injury is followed by a proliferation of the medial smooth muscle cells,after which many of them migrate into the intima through fenestrae inthe internal elastic lamina and proliferate to form a neointimal lesion.

Vascular stenosis can be detected and evaluated using angiographic orsonographic imaging techniques [Evans, R. G., JAMA 265:2382 (1991)] andis often treated by percutaneous transluminal coronary angioplasty(balloon catheterization). Within a few months following angioplasty,however, the blood flow is reduced in approximately 30-40 percent ofthese patients as a result of restenosis caused by a response tomechanical vascular injury suffered during the angioplasty procedure, asdescribed above. [Pepine, C., Circulation 81:1753 (1990); Hardoff, R.,J. Am. Coll. Cardiol. 15 1486 (1990)].

In an attempt to prevent restenosis or reduce intimal smooth muscle cellproliferation following angioplasty, numerous pharmaceutical agents havebeen employed clinically, concurrent with or following angioplasty. Mostpharmaceutical agents employed in an attempt to prevent or reduce theextent of restenosis have been unsuccessful. The following listidentifies several of the agents for which favorable clinical resultshave been reported: lovastatin [Sahni, R., Circulation 80 (Suppl.) 65(1989); Gellman, J., J. Am. Coll. Cardiol. 17:251 (1991)]; thromboxaneA₂ synthetase inhibitors such as DP-1904 [Yabe, Y., Circulation 80(Suppl.) 260 (1989)]; eicosapentanoic acid [Nye, E., Aust. N. Z. J. Med.20:549 (1990)]; ciprostene (a prostacyclin analog) [Demke, D., Brit. J.Haematol 76 (Suppl.): 20 (1990); Darius, H., Eur. Heart J. 12 (Suppl.):26 (1991)]; trapidil (a platelet derived growth factor) [Okamoto, S.,Circulation 82 (Suppl.): 428 (1990)]; angiotensin converting enzymeinhibitors [Gottlieb, N., J. Am. Coll. Cardiol. 17 (Suppl. A): 181A(1991)]; and low molecular weight heparin [de Vries, C., Eur. Heart J.12 (Suppl.): 386 (1991)].

In an attempt to develop better agents for preventing or reducing smoothmuscle proliferation and intimal thickening, the use of balloon catheterinduced arterial injury in a variety of mammals has been developed as astandard model of vascular injury that will lead to intimal thickeningand eventual vascular narrowing. [Chevru, A., Surg. Gynecol. Obstet.171:443 (1990); Fishman, J., Lab. Invest. 32:339 (1975); Haudenschild,C., Lab. Invest. 41:407 (1979); Clowes, A. W., Lab. Invest. 49:208(1983); Clowes, A. W., J. Cardiovas. Pharm. 14:S12 (1989); and Ferns, G.A., Science 253:1129 (1991)]. Many compounds have been evaluated in thisstandard animal model. The immunosuppressive agent cyclosporin A hasbeen evaluated and has produced conflicting results. Jonasson reportedthat cyclosporin A caused an inhibition of the intimal proliferativelesion following arterial balloon catheterization in vivo, but did notinhibit smooth muscle cell proliferation in vitro. [Jonasson, L., Proc.Natl. Acad. Sci. 85:2303 (1988)]. Ferns, however reported that whende-endothelilized rabbits were treated with cyclosporin A, nosignificant reduction of intimal proliferation was observed in vivo.Additionally, intimal accumulations of foamy macrophages, together witha number of vacuolated smooth muscle cells in the region adjacent to theinternal elastic lamina were observed, indicating that cyclosporin A maymodify and enhance lesions that form at the sites of arterial injury.[Ferns, G. A., Circulation 80 (Supp): 184 (1989); Ferns, G., Am. J.Path. 137:403 (1990)].

Rapamycin, a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus [U.S. Pat. No. 3,929,992] has been shown to prevent theformation of humoral (IgE-like) antibodies in response to an albuminallergic challenge [Martel, R., Can. J. Physiol. Pharm. 55:48 (1977)],inhibit murine T-cell activation [Staruch, M., FASEB 3:3411 (1989)],prolong survival time of organ grafts in histoincompatible rodents[Morris, R., Med. Sci. Res. 17:877 (1989)], and inhibit transplantationrejection in mammals [Calne, R., European Patent Application 401,747].Rapamycin blocks calcium-dependent, calcium-independent,cytokine-independent and constitutive T and B cell division at the G1-Sinterface. Rapamycin inhibits gamma-interferon production induced byI1-1 and also inhibits the gamma-interferon induced expression ofmembrane antigen. [Morris, R. E., Transplantation Rev. 6:39 (1992)]. Theuse of rapamycin in preventing coronary graft atherosclerosis (CGA) inrats has been disclosed by Meiser [J. Heart Lung Transplant 9:55(1990)]. Arterial thickening following transplantation, known as CGA, isa limiting factor in graft survival that is caused by a chronicimmunological response to the transplanted blood vessels by thetransplant recipient's immune system. [Dec. G, Transplantation Proc.23:2095 (1991) and Dunn, M. Lancet 339:1566 (1992)]. The disclosedinvention is distinct from the use of rapamycin for preventing CGA, inthat CGA does not involve injury to the recipients own blood vessels; itis a rejection type response. The disclosed invention is related tovascular injury to native blood vessels. The resulting intimal smoothmuscle cell proliferation dose not involve the immune system, but isgrowth factor mediated. For example, arterial intimal thickening afterballoon catheter injury is believed to be caused by growth factor (PGDF,bFGF, TGFb, IL-1 and others)-induced smooth muscle cell proliferationand migration. [Ip, J. H., J. Am. Coll. Cardiol 15:1667 (1990)]. Fernshas also shown that the immune response is not involved in arterialintimal thickening following balloon catheterization, as he found thatthere was no difference in intimal thickening between arteries fromathymic nude rats (rats lacking T-cells) and normal rats after ballooncatheterization [Am. J. Pathol. 138:1045 (1991)].

DESCRIPTION OF THE INVENTION

This invention provides a method of preventing or treatinghyperproliferative vascular disease in a mammal in need thereof byadministering an antiproliferative effective amount of rapamycin to saidmammal orally, parenterally, intravascularly, intranasally,intrabronchially, transdermally, rectally, or via a vascular stentimpregnated with rapamycin.

As such, rapamycin is useful in treating intimal smooth muscle cellhyperplasia, restenosis, and vascular occlusion in a mammal,particularly following either biologically or mechanically mediatedvascular injury, or under conditions that would predispose a mammal tosuffering such a vascular injury. Biologically mediated vascular injuryincludes, but is not limited to injury attributed to infectiousdisorders including endotoxins and herpes viruses such ascytomegalovirus; metabolic disorders such as atherosclerosis; andvascular injury resulting from hypothermia, and irradiation.Mechanically mediated vascular injury includes, but is not limited tovascular injury caused by catheterization procedures or vascularscraping procedures such as percutaneous transluminal coronaryangioplasty; vascular surgery; transplantation surgery; laser treatment;and other invasive procedures which disrupt the integrity of thevascular intima or endothelium. Rapamycin is also useful in preventingintimal smooth muscle cell hyperplasia, restenosis, and vascularocclusion resulting from mechanically mediated injury. In particular,for the prevention of restenosis following a percutaneous transluminalcoronary angioplasty procedure.

Treating includes retarding the progression, arresting the development,as well as palliation. Preventing includes inhibiting the development ofand prophylacticly preventing of hyperproliferative vascular disease ina susceptible mammal.

This invention also provides a method of using a combination ofrapamycin and mycophenolic acid for the same utilities described above.Mycophenolic acid, an antiproliferative antimetabolite, inhibits inosinemonophosphate dehydrogenase and guanosine monophosphate synthetase,enzymes in the de novo purine biosynthetic pathway. This results in aninhibition of DNA synthesis which causes an accumulation of cells at theG1-S interface. Other combinations containing rapamycin that are usefulfor preventing or treating hyperproliferative vascular disease will beapparent to one skilled in the art. These include, but are not limitedto, using rapamycin in combination with other antiproliferativeantimetabolites.

The effect of rapamycin on hyperproliferative vascular disease wasestablished in an in vitro and an in vivo standard pharmacological testprocedure that emulates the hyperproliferative effects observed inmammals that are undergoing intimal smooth muscle proliferation and aretherefore developing restenosis. Cycloporin A was also evaluated inthese test procedures for the purpose of comparison. The combination ofrapamycin and mycophenolic acid was evaluated in the in vivo testprocedure. The procedures and the results obtained are described below.

Rapamycin and cyclosporin A were evaluated in an in vitro standardpharmacological test procedure which emulates the intimal smooth musclecell proliferation observed following vascular injury. Results wereobtained by measuring DNA and protein synthesis in rat smooth musclecells that have been stimulated with a growth factor such as fetal calfserum or a hypertrophic mitogen, such as angiotensin II. The followingbriefly describes the procedure that was used. Rat smooth muscle cellswere maintained in a 1:1 mixture of defined Eagle's medium (DEM) andHam's F12 medium with 10% fetal calf serum, penicillin (100 U/mL),streptomycin (100 mg/mL) and 25 mL Hepes at pH 7.4. Cells were incubatedat 37° C. in a humidified atmosphere of 5% CO₂ with media changes every2-3 days. Each compound tested was diluted with an appropriate vehicleto obtain a 1 mM stock solution. Ethanol was used as the vehicle forrapamycin and 20% tween 80 in ethanol was the vehicle for cyclosporin A.Test concentrations of drug were obtained by diluting appropriateconcentrations of stock solution with serum free media. The smoothmuscle cell culture was maintained in a defined serum free mediacontaining 1:1 DEM and Ham's F12 medium, insulin (5×10⁻⁷ M), transferrin(5 μg/mL), and ascorbate (0.2 mM) for 72 hours before testing in amulti-well plate. After the 72 hour period, an appropriate quantity ofstock solution containing either rapamycin or cyclosporin A was added tothe smooth muscle cell culture and media mixture. After a 24 hours theappropriate growth factor was added. For the measurement of DNAsynthesis, ³ H-thymidine was added at 12 hours after the growth factorwas added, and the cells were harvested at 36 hours. For the measurementof protein synthesis, ³ H-leucine was added at 14 hours after the growthfactor was added, and the cells were harvested at 18 hours. The amountof incorporated radioactive label was measured on a scintillationcounter.

The following table shows the results obtained for rapamycin on DNA andprotein synthesis in smooth muscle cells that were stimulated with 10%fetal calf serum, as measured by incorporation of tritiated thymidine orleucine into smooth muscle cells. The amount of tritiated labelincorporated by the smooth muscle cells that were treated with mediaonly was normalized to 100%, and the results for cells treated withfetal calf serum or fetal calf serum plus the test compound areexpressed as a percent comparison with the cells treated with mediaonly.

    ______________________________________                                        EFFECT OF RAPAMYCIN ON DNA AND PROTEIN SYNTHESIS                              IN SMOOTH CELLS STIMULATED WITH FETAL CALF SERUM*                                           .sup.3 H-Thymidine                                                                     .sup.3 H-Leucine                                                     Incorporation                                                                          Incorporation                                                        (% of Media)                                                                           (% of Media)                                           ______________________________________                                        Media           100%       100%                                               FCS             495%       174%                                               1000 nM RAP + FCS                                                                             136%        95%                                                100 nM RAP + FCS                                                                             172%        91%                                                10 nM RAP + FCS                                                                              204%        74%                                                 1 nM RAP + FCS                                                                              403%       106%                                               ______________________________________                                         *Abbreviations: RAP = rapamycin; Media = defined serum free media; and FC     = 10% fetal calf serum.                                                  

The following table shows the results obtained for rapamycin on proteinsynthesis in smooth muscle cells that were stimulated with 10⁻⁶ nMangiotensin II, as measured by incorporation of tritiated leucine intosmooth muscle cells. The amount of tritiated label incorporated by thesmooth muscle cells that were treated with media only were normalized to100%, and the results for cells treated with angiotensin or angiotensinplus the test compound are expressed as a percent comparison with thecells treated with media only.

    ______________________________________                                        EFFECT OF RAPAMYCIN ON PROTEIN SYNTHESIS                                      IN SMOOTH CELLS STIMULATED WITH ANGIOTENSIN II*                                            .sup.3 H-Leucine Incorporation                                                (% of Media)                                                     ______________________________________                                        Media          100%                                                           ANG            159%                                                           1000 nM RAP + ANG                                                                            53%                                                             100 nM RAP + ANG                                                                            57%                                                             10 nM RAP + ANG                                                                             61%                                                              1 nM RAP + ANG                                                                             60%                                                            ______________________________________                                         *Abbreviations: RAP = rapamycin; Media = defined serum free media; and AN     = 10.sup.-6 nM angiotensin II.                                           

The results of the standard in vitro test procedure showed thatrapamycin had a pronounced antiproliferative effect in the presence ofFCS and an anti-hypertrophic effect in the presence of angiotensin II.Following vascular injury, DNA and protein synthesis of smooth musclecells are necessary for the development of restenosis to occur. Theseresults showed that rapamycin inhibited both DNA and protein synthesisin stimulated smooth muscle cells. An antiproliferative effect was alsoobserved with cyclosporin A; however, at 1000 nM, cyclosporin A wascytotoxic and not merely cytostatic. At 1000 nM, cyclosporin A causedlysis of the smooth muscle cells as evidenced by the presence of lacticacid dehydrogenase in the supernatant of the cell culture. Similartoxicity to smooth muscle cells was not observed for rapamycin.

Rapamycin, rapamycin plus mycophenolic acid, and cyclosporin A wereevaluated in an in vivo standard pharmacological test procedure thatemulates the vascular injury suffered and restenosis (hat developsfollowing percutaneous transluminal coronary angioplasty in humans. Theability of a test compound to inhibit restenosis was determined bycomparing intimal thickening in mammals treated with test compoundfollowing balloon catheterization versus intimal thickening in untreatedcontrol mammals after the same test procedure. [Chevru, A., Surg.Gynecol. Obstet. 171:443 (1990); Fishman, J., Lab. Invest. 32:339(1975); Haudenschild, C., Lab. Invest. 41:407 (1979); Clowes, A. W.,Lab. Invest. 49:208 (1983); Clowes, A. W., J. Cardiovas. Pharm. 14:S12(1989); and Ferns, G. A., Science 253:1129 (1991)]. The followingbriefly describes the procedure that was used. The left carotid arteriesof male Sprague-Dawley rats were injured with an inflated 2 Fr ballooncatheter. During a 14 day postoperative period, these rats were dividedinto groups and treated daily with rapamycin (1.5 mg/kg; i.p.),rapamycin plus mycophenolic acid (1.5 mg/kg; i.p. +40 mg/kg; p.o.), orcyclosporin A (3 mg/kg; i.p.). Treatment was administered on days 0 to13 postoperatively. Additionally, one group each also received rapamycin(6 mg/kg/day; i.p.) or cyclosporin A (40 mg/kg/day; i.p.) for two dayspostoperatively, and then received no treatment for the next 12 days. Anuntreated group was used an injured control to establish the amount ofintimal growth in the absence of treatment. The right carotid was usedas an uninjured control in all groups. After the 14-day period, the ratswere sacrificed, the carotids removed. The mean areas of the intima andblood vessel wall were measured by morphometry. Results are expressed asan intima percent which can be expressed according to the followingformula: ##EQU1##

The following table shows the results that were obtained.

    ______________________________________                                        EFFECT OF RAPAMYCIN ON INTIMAL THICKENING IN                                  INJURED CAROTIID ARTERIES (DAY 14)*                                           Test Group       Intima Percent ± S.E.                                     ______________________________________                                        Uninjured Control                                                                              0.00 ± 0.00                                               Untreated Injured Control                                                                       33.3 ± 19.66                                             RAP (1.5 mg/kg - 14 days)                                                                      6.78 ± 4.69                                               RAP (6 mg/kg - 2 days)                                                                         16.56 ± 6.22                                              RAP + MPA (14 days)                                                                            1.6 ± 3.5                                                 CsA (3 mg/kg - 14 days)                                                                        26.46 ± 27.42                                             CsA (40 mg/kg - 2 days)                                                                        31.14 ± 20.66                                             ______________________________________                                         *Abbreviations RAP = rapamycin; MPA = mycophenolic acid; and CsA =            cyclosporin A.                                                           

These results show that treatment with rapamycin (1.5 mg/kg for 14 days)resulted in an 80% decrease in the mean percentage intimal thickeningcompared with the untreated injured control group. Similarly, treatmentwith the combination of rapamycin and mycophenolic acid produced almosta complete inhibition of intimal thickening (95% reduction in intimalthickening compared with untreated injured control). Cyclosporin Afailed to produce any meaningful reduction in intimal thickening.

Similar results were obtained when rapamycin was evaluated at differentdoses in the above in vivo standard pharmacological test procedure thatemulates the vascular injury that occurs following a percutaneoustransluminal coronary angioplasty procedure in humans. Rapamycin wasadministered on postoperative days 0-13, and examination by morphometrywas performed on day 14. Rapamycin, at a dose of 1.5 and 3 mg/kgsignificantly arrested the development of restenosis as measured by theintima percent 14 days after balloon catheterization, whereas restenosiswas clearly observed in the untreated injured control group. Theseresults are summarized in the table below.

    ______________________________________                                        EFFECT OF RAPAMYCIN ON INTIMAL THICKENING IN                                  INJURED CAROTID ARTERIES (DAY 14)                                             Group     Dose     Treatment Days                                                                            Intima Percent ± S.E.                       ______________________________________                                        Uninjured Control              0.00 ± 0.00                                 Untreated Injured              44.51 ± 5.03                                Control                                                                       Rapamycin   6 mg/kg                                                                              0-13        30.92 ± 4.06                                Rapamycin   3 mg/kg                                                                              0-13        22.68 ± 6.28                                Rapamycin 1.5 mg/kg                                                                              0-13        21.89 ± 4.2                                 ______________________________________                                    

The results of the in vitro and in vivo standard test proceduresdemonstrate that rapamycin and rapamycin in combination withmycophenolic acid are useful in treating hyperproliferative vasculardisease.

As such, rapamycin is useful in treating intimal smooth muscle cellhyperplasia, restenosis, and vascular occlusion in a mammal,particularly following either biologically or mechanically mediatedvascular injury, or under conditions that would predispose a mammal tosuffering such a vascular injury. Biologically mediated vascular injuryincludes, but is not limited to injury attributed to infectiousdisorders including endotoxins and herpes viruses such ascytomegalovirus; metabolic disorders such as atherosclerosis; andvascular injury resulting from hypothermia, and irradiation.Mechanically mediated vascular injury includes, but is not limited tovascular injury caused by catheterization procedures or vascularscraping procedures such as percutaneous transluminal coronaryangioplasty; vascular surgery; transplantation surgery; laser treatment;and other invasive procedures which disrupt the integrity of thevascular intima or endothelium.

Rapamycin and rapamycin plus mycophenolic acid were also evaluated in amodification of the in vivo test procedure described above. In themodified test procedure, treatment with rapamycin or rapamycin plusmycophenolic acid were stopped on day 14, as above, but the animals werenot sacrificed immediately. Intimal thickening was observed when theanimals were sacrificed 1, 2, 4 weeks, and 44 days after treatment hadbeen stopped. Microscopic analysis showed that endothelium regenerationhad not occurred during the two week treatment period. For example, 44days after undergoing balloon catheterization procedure of the carotidartery, untreated injured control rats had an intima percent (±S.E.) of62.85±3.63, and rats treated with rapamycin+mycophenolic acid (1.5/40mg/kg) on postoperative days 0-13 had an intima percent (±S.E.) of50.39±2.58. Better results were not obtained when the same regimen wasadministered on days 0-30 (intima percent (±S.E.) of 53.55±2.85).Following cessation of treatment with rapamycin or rapamycin plusmycophenolic acid intimal proliferation, that was previously suppressed,was able to occur. These results are consistent with the results shownin the table above, in which treatment for 2 days with rapamycinfollowed by 12 days of no treatment inhibited intimal thickening to alesser degree than treatment with rapamycin for 14 days. These resultsare expected, as in the absence on an integral endothelial layer, theintimal smooth muscle cells will proliferate. It has been shown thatintimal smooth muscle cell growth does not have an inhibitory effect onnormal endothelial regeneration, and that intimal smooth muscle cellproliferation ceases when the endothelial layer is established. [Reidy,M., Lab. Invest. 59:36 (1988); Chevru, A., Surg. Gynecol. Obstet.171:443 (1990); Fishman, J., Lab. Invest. 32:339 (1975); Haudenschild,C., Lab. Invest. 41:407 (1979)]. As such, treatment with rapamycin orrapamycin in combination with mycophenolic acid should be employed solong as the beneficial effect is seen. As the degree of restenosis canbe monitored by angiographic and sonographic techniques, the dosagenecessary to sustain the opened vessels can be adjusted.

To evaluate the ability of rapamycin and rapamycin plus mycophenolicacid to prevent restenosis following an angioplasty procedure, rapamycinwas evaluated in the same in vivo standard pharmacological testprocedure for restenosis that was described above, except that treatmentwith rapamycin began three days before (day -3) the angioplastyprocedure was performed. The following table shows the results obtainedon day 14 following balloon catheterization of the carotid artery on day0. Results for treatment from day 3 to 13 are also provided.

    ______________________________________                                        EFFECT OF RAPAMYCIN ON INTIMAL THICKENING IN                                  INJURED CAROTID ARTERIES (DAY 14)                                             Group     Dose     Treatment Days                                                                            Intima Percent ± S.E.                       ______________________________________                                        Uninjured Control              0.00 ± 0.00                                 Untreated Injured              44.51 ± 5.03                                Control                                                                       Rapamycin 1.5 mg/kg                                                                               -3-13*     9.85 ± 1.15                                 Rapamycin 1.5 mg/kg                                                                              -3-3        30.7 ± 6.67                                 Rapamycin 1.5 mg/kg                                                                              -3-0        37.31 ± 4.33                                Rapamycin 1.5 mg/kg                                                                                 3-13     44.38 ± 5.49                                ______________________________________                                         *Treatment from three days preballoon catheterization to day 13 days          postcatheterization.                                                     

The results in the table above show that rapamycin prevented thedevelopment of restenosis following a balloon angioplasty procedure ofthe carotid artery, when rapamycin was administered from three dayspre-angioplasty until day 13. Treatment from day minus 3 until day 3 orday 0 afforded a lesser degree of prevention, and treatment from day 3to day 13 did not prevent restenosis.

The effect of rapamycin plus mycophenolic acid (MPA) was also evaluatedin the angioplasty standard pharmacological test procedure. The tablebelow shows the results obtained where rats underwent a ballooncatheterization procedure of the carotid artery on day 0, and weresacrificed and examined morphometrically on day 44. The treatmentregimen is described in the table.

    ______________________________________                                        EFFECT OF RAPAMYCIN + MPA ON INTIMAL THICKENING                               IN INJURED CAROTID ARTERIES (DAY 44)                                                                 Treatment Intima                                       Group       Dose       Days      Percent ± S.E.                            ______________________________________                                        Uninjured Control                0.00 ± 0.00                               Untreated Injured                62.85 ± 3.63                              Control                                                                       Rapamycin + MPA                                                                           40/1.5 mg/kg                                                                             0-13      50.39 ± 2.58                              Rapamycin + MPA                                                                           40/1.5 mg/kg                                                                             0-30      53.55 ± 2.85                              Rapamycin + MPA                                                                           40/1.5 mg/kg                                                                             -3-13     18.76 ± 10.6                              ______________________________________                                    

These results show that treatment with rapamycin and mycophenolic acidfrom day minus 3 to day 13 did effectively prevent restenosis at day 44,whereas the regimens which did not include drug administration beforethe angioplasty procedure did not effectively prevent restenosis at day44.

Similar results were obtained when rat thoracic aortas were subjected toa balloon catheterization procedure, as described above, on day 0. Therats were either sacrificed and examined on day 14 or on day 44. Theresults obtained with rapamycin and rapamycin plus mycophenolic acid(MPA) are shown in the table below.

    ______________________________________                                        EFFECT OF RAPAMYCIN AND RAPAMYCIN + MPA ON                                    THICKENING IN INJURED THORACIC AORTAS                                                                Treatment Intima                                       Group       Dose       Days      Percent ± S.E.                            ______________________________________                                        Day 14 results                                                                Uninjured Control                0.00 ± 0.00                               Untreated Injured                15.52 ± 2.99                              Control                                                                       Rapamycin + MPA                                                                           40/1.5 mg/kg                                                                             -3-13     0.00 ± 0.00                               Day 44 Results                                                                Uninjured Control                0.00 ± 0.00                               Untreated Injured                28.76 ± 6.52                              Control                                                                       Rapamycin   1.5 mg/kg  -3-13     0.00 ± 0.00                               Rapamycin + MPA                                                                           40/1.5 mg/kg                                                                             -3-13     8.76 ± 3.34                               ______________________________________                                    

The results in the table above show that treatment with rapamycin from 3days preoperatively until 13 days postoperatively completely preventedthe development of restenosis 44 days after a balloon catheterization ofthe thoracic aorta. Using the same treatment regimen, rapamycin plusmycophenolic acid completely prevented restenosis 14 days after ballooncatheterization and significantly prevented restenosis 44 days followingballoon catheterization.

Similarly, day minus 3 to day 13 treatment with rapamycin plusmycophenolic acid completely prevented restenosis 14 days after ballooncatheterizaton of the abdominal aortas in rats. These results are shownin the table below.

    ______________________________________                                        EFFECT OF RAPAMYCIN + MPA ON INTIMAL THICKENING                               IN INJURED ABDOMINAL AORTAS (DAY 14)                                                                 Treatment Intima                                       Group       Dose       Days      Percent ± S.E.                            ______________________________________                                        Uninjured Control                0.00 ± 0.00                               Untreated Injured                10.17 ± 2.42                              Control                                                                       Rapamycin + MPA                                                                           40/1.5 mg/kg                                                                             -3-13     0.00 ± 0.00                               ______________________________________                                    

The results in the tables above show that rapamycin, alone or incombination with mycophenolic acid, is useful in preventing restenosisfollowing invasive procedures that disrupt the vascular endotheliallining, such as percutaneous transluminal coronary angioplasty, vascularcatheterization, vascular scraping, vascular surgery, or laser treatmentprocedures. These data also show that the administration of rapamycin,alone or in combination with mycophenolic acid, from 3 dayspre-catheterization to 13 days post-catheterization, allowed theendothelium to heal, while preventing intimal smooth muscle cellproliferation. That intimal proliferation did not occur 31 days afteradministration with rapamycin, alone or in combination with mycophenolicacid, had been stopped, demonstrates that the endothelial layer hadregenerated, as intimal proliferation stops after the reestablishment ofthe endothelial layer. The reestablishment of an intact endotheliallayer was confirmed by microscopic examination of the previouslycatheterized arteries after removal at 44 days.

From the data above, it is particularly preferred that treatment beginwith rapamycin or rapamycin plus mycophenolic acid before the procedureis performed, and that treatment should continue after the procedure hasbeen performed. The length of treatment necessary to prevent restenosiswill vary from patient to patient. For percutaneous transluminalangioplasty procedures, it is preferred that treatment be administeredfrom 3 or more days before the procedure and continuing for 8 or moredays after the procedure. It is more preferred that administration willbe for 3 or more days before the angioplasty procedure and continuingfor 13 or more days after the procedure. The same administrationprotocol is applicable when rapamycin, alone or in combination withmycophenolic acid, is used to prevent restenosis following vascularcatheterization, vascular scraping, vascular surgery, or laser treatmentprocedures.

When rapamycin is employed alone or in combination with mycophenolicacid in the prevention or treatment of hyperproliferative vasculardisease, it can be formulated neat or with a pharmaceutical carrier to amammal in need thereof. The pharmaceutical carrier may be solid orliquid.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenated,hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

Rapamycin, alone or in combination with mycophenolic acid, may beadministered rectally in the form of a conventional suppository. Foradministration by intranasal or intrabronchial inhalation orinsufflation, the compounds of this invention may be formulated into anaqueous or partially aqueous solution, which can then be utilized in theform of an aerosol. Rapamycin, alone or in combination with mycophenolicacid, may also be administered transdermally through the use of atransdermal patch containing the active compound and a carrier that isinert to the active compound, is non toxic to the skin, and allowsdelivery of the agent for systemic absorption into the blood stream viathe skin. The carrier may take any number of forms such as creams andointments, pastes, gels, and occlusive devices. The creams and ointmentsmay be viscous liquid or semisolid emulsions of either the oil-in-wateror water-in-oil type. Pastes comprised of absorptive powders dispersedin petroleum or hydrophilic petroleum containing the active ingredientmay also be suitable. A variety of occlusive devices may be used torelease the active ingredient into the blood stream such as asemipermiable membrane covering a reservoir containing the activeingredient with or without a carrier, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

Rapamycin, alone or in combination with mycophenolic acid can beadministered intravascularly or via a vascular stent impregnated withrapamycin, alone or in combination with mycophenolic acid, duringballoon catheterization to provide localized effects immediatelyfollowing injury.

Rapamycin, alone or in combination with mycophenolic acid, may beadministered topically as a solution, cream, or lotion by formulationwith pharmaceutically acceptable vehicles containing 0.1-5 percent,preferably 2%, of active compound.

The dosage requirements vary with the particular compositions employed,the route of administration, the severity of the symptoms presented andthe particular subject being treated. Based on the results obtained inthe standard pharmacological test procedures, projected dailyintravenous dosages of rapamycin, when administered as the sole activecompound or in combination with mycophenolic acid, would be 0.001-25mg/kg, preferably between 0.005-10 mg/kg, and more preferably between0.01-5 mg/kg. Projected daily oral dosages of rapamycin, whenadministered as the sole active compound or in combination withmycophenolic acid, would be 0.005-50 mg/kg, preferably between 0.01-25mg/kg, and more preferably between 0.05-10 mg/kg. Projected dailyintravenous dosages of mycophenolic acid, when used in combination withrapamycin, would be 0.5-75 mg/kg and preferably between 5-50 mg/kg.Projected daily oral dosages of mycophenolic acid, when used incombination with rapamycin, would be 1-75 mg/kg and preferably between10-50 mg/kg.

Treatment will generally be initiated with small dosages less than theoptimum dose of the compound. Thereafter the dosage is increased untilthe optimum effect under the circumstances is reached; precise dosagesfor oral, parenteral, intravascular, intranasal, intrabronchial,transdermal, or rectal administration will be determined by theadministering physician based on experience with the individual subjecttreated. In general, rapamycin is most desirably administered at aconcentration that will generally afford effective results withoutcausing any harmful or deleterious side effects, and can be administeredeither as a single unit dose, or if desired, the dosage may be dividedinto convenient subunits administered at suitable times throughout theday.

What is claimed is:
 1. A method of treating a hyperproliferativevascular disease selected from the group consisting of intimal smoothmuscle cell proliferation, restenosis, and vascular occlusion, whereinthe intimal smooth muscle cell proliferation, restenosis, or vascularocclusion is caused by an infectious disorder, hypothermia, orirradiation, which comprises administering an antiproliferativeeffective amount of rapamycin to said mammal orally, parenterally,intravascularly, intranasally, intrabronchially, transdermally,rectally, or via a vascular stent impregnated with rapamycin.
 2. Amethod of treating a hyperproliferative vascular disease selected fromthe group consisting of intimal smooth muscle cell proliferation,restenosis, and vascular occlusion, wherein the intimal smooth musclecell proliferation, restenosis, or vascular occlusion is caused byvascular catheterization, vascular scraping, percutaneoustransluminal/coronary angioplasty, vascular surgery, or laser treatment,which comprises, administering an antiproliferative effective amount ofrapamycin to said mammal orally, parenterally, intravascularly,intranasally, intrabronchially, transdermally, rectally, or via avascular stent impregnated with rapamycin.